Ebola Virus-Specific Neutralizing Antibody Persists at High Levels in Survivors 2 Years After Resolution of Disease in a Sierra Leonean Cohort.

Ebola virus (EBOV) infection results in Ebola virus disease (EVD), an often severe disease with a nonspecific presentation. Since its recognition, periodic outbreaks of EVD continue to occur in sub-Saharan Africa. The 2013-2016 West African EVD outbreak was the largest recorded, resulting in a substantial cohort of EVD survivors with persistent health complaints and variable immune responses. In this study, we characterize humoral immune responses in EVD survivors and their contacts in Eastern Sierra Leone. We found high levels of EBOV IgG in EVD survivors and lower yet substantial antibody levels in household contacts, suggesting subclinical transmission. Neutralizing antibody function was prevalent but variable in EVD survivors, raising questions about the durability of immune responses from natural infection with EBOV. Additionally, we found that certain discrete symptoms-ophthalmologic and auditory-are associated with EBOV IgG seropositivity, while an array of symptoms are associated with the presence of neutralizing antibody.

Genome-wide association study identifies human genetic variants associated with fatal outcome from Lassa fever.

Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis.

Novel Tools for Lassa Virus Surveillance in Peri-domestic Rodents.

Background: Lassa fever (LF) is a rodent-borne disease endemic to West Africa. In the absence of licensed therapeutics or vaccines, rodent exclusion from living spaces remains the primary method of preventing LF. Zoonotic surveillance of Lassa virus (LASV), the etiologic agent of LF, can assess the burden of LASV in a region and guide public health measures against LF. Methods: In this study, we adapted commercially available LASV human diagnostics to assess the prevalence of LASV in peri-domestic rodents in Eastern Sierra Leone. Small mammal trapping was conducted in Kenema district, Sierra Leone between November 2018-July 2019. LASV antigen was detected using a commercially available LASV NP antigen rapid diagnostic test. LASV IgG antibodies against LASV nucleoprotein (NP) and glycoprotein (GP) were tested by adapting a commercially available semi-quantitative enzyme linked immunosorbent assay (ELISA) for detection of mouse-related and rat-related species IgG. Findings: Of the 373 tested specimens, 74 (20%) tested positive for LASV antigen. 40 (11%) specimens tested positive for LASV NP IgG, while an additional 12 (3%) specimens only tested positive for LASV GP IgG. Simultaneous antigen presence and IgG antibody presence was linked in Mastomys sp. specimens (p < 0.01), but not Rattus sp. specimens (p = 1). Despite the link between antigen presence and IgG antibody presence in Mastomys sp., the strength of antigen response did not correlate with the strength of IgG response to either GP IgG or NP IgG. Interpretation: The tools developed in this study can aid in the generation of valuable public health data for rapid field assessment of LASV burden during outbreak investigations and general LASV surveillance. Funding: Funding for this work was supported by the National Institute of Allergy and Infectious Diseases National Institute of Health, Department of Health and Human Services under the following grants: International Collaboration in Infectious Disease Research on Lassa fever and Ebola - ICIDR - U19 AI115589, Consortium for Viral Systems Biology - CViSB - 5U19AI135995, West African Emerging Infectious Disease Research Center - WARN-ID - U01AI151812, West African Center for Emerging Infectious Diseases: U01AI151801.

A prospective, multi-site, cohort study to estimate incidence of infection and disease due to Lassa fever virus in West African countries (the Enable Lassa research programme)-Study protocol.

BACKGROUND: Lassa fever (LF), a haemorrhagic illness caused by the Lassa fever virus (LASV), is endemic in West Africa and causes 5000 fatalities every year. The true prevalence and incidence rates of LF are unknown as infections are often asymptomatic, clinical presentations are varied, and surveillance systems are not robust. The aim of the Enable Lassa research programme is to estimate the incidences of LASV infection and LF disease in five West African countries. The core protocol described here harmonises key study components, such as eligibility criteria, case definitions, outcome measures, and laboratory tests, which will maximise the comparability of data for between-country analyses. METHOD: We are conducting a prospective cohort study in Benin, Guinea, Liberia, Nigeria (three sites), and Sierra Leone from 2020 to 2023, with 24 months of follow-up. Each site will assess the incidence of LASV infection, LF disease, or both. When both incidences are assessed the LASV cohort (nmin = 1000 per site) will be drawn from the LF cohort (nmin = 5000 per site). During recruitment participants will complete questionnaires on household composition, socioeconomic status, demographic characteristics, and LF history, and blood samples will be collected to determine IgG LASV serostatus. LF disease cohort participants will be contacted biweekly to identify acute febrile cases, from whom blood samples will be drawn to test for active LASV infection using RT-PCR. Symptom and treatment data will be abstracted from medical records of LF cases. LF survivors will be followed up after four months to assess sequelae, specifically sensorineural hearing loss. LASV infection cohort participants will be asked for a blood sample every six months to assess LASV serostatus (IgG and IgM). DISCUSSION: Data on LASV infection and LF disease incidence in West Africa from this research programme will determine the feasibility of future Phase IIb or III clinical trials for LF vaccine candidates.

Seroprevalence of anti-Lassa Virus IgG antibodies in three districts of Sierra Leone: A cross-sectional, population-based study.

BACKGROUND: Lassa virus (LASV), the cause of the acute viral hemorrhagic illness Lassa fever (LF), is endemic in West Africa. Infections in humans occur mainly after exposure to infected excrement or urine of the rodent-host, Mastomys natalensis. The prevalence of exposure to LASV in Sierra Leone is crudely estimated and largely unknown. This cross-sectional study aimed to establish a baseline point seroprevalence of IgG antibodies to LASV in three administrative districts of Sierra Leone and identify potential risk factors for seropositivity and LASV exposure. METHODOLOGY AND PRINCIPAL FINDINGS: Between 2015 and 2018, over 10,642 participants from Kenema, Tonkolili, and Port Loko Districts were enrolled in this cross-sectional study. Previous LASV and LF epidemiological studies support classification of these districts as "endemic," "emerging," and "non-endemic", respectively. Dried blood spot samples were tested for LASV antibodies by ELISA to determine the seropositivity of participants, indicating previous exposure to LASV. Surveys were administered to each participant to assess demographic and environmental factors associated with a higher risk of exposure to LASV. Overall seroprevalence for antibodies to LASV was 16.0%. In Kenema, Port Loko, and Tonkolili Districts, seroprevalences were 20.1%, 14.1%, and 10.6%, respectively. In a multivariate analysis, individuals were more likely to be LASV seropositive if they were living in Kenema District, regardless of sex, age, or occupation. Environmental factors contributed to an increased risk of LASV exposure, including poor housing construction and proximity to bushland, forested areas, and refuse. CONCLUSIONS AND SIGNIFICANCE: In this study we determine a baseline LASV seroprevalence in three districts which will inform future epidemiological, ecological, and clinical studies on LF and the LASV in Sierra Leone. The heterogeneity of the distribution of LASV and LF over both space, and time, can make the design of efficacy trials and intervention programs difficult. Having more studies on the prevalence of LASV and identifying potential hyper-endemic areas will greatly increase the awareness of LF and improve targeted control programs related to LASV.

Expansion of CD8+ T cell population in Lassa virus survivors with low T cell precursor frequency reveals durable immune response in most survivors.

INTRODUCTION: Lassa virus is a priority pathogen for vaccine research and development, however the duration of cellular immunity and protection in Lassa fever (LF) survivors remains unclear. METHODS: We investigated Lassa virus specific CD8+ T cell responses in 93 LF survivors. Peripheral blood mononuclear cells from these individuals were infected with recombinant vesicular stomatitis virus encoding Lassa virus antigens and virus specific T cell responses were measured after 18-hour incubation. Participants who had undetectable CD8+ T cell response underwent further analysis using a 10-day T cell proliferation assays to evaluate for low T cell precursor frequency. RESULTS: Forty-five of the 93 LF survivors did not have a Lassa virus specific CD8+ T cell response. Of those with responses and a known date of onset of LF (N = 11), 9 had LF within the last ten years. Most participants without a measurable CD8+ T cell response were more than 10 years removed from a clinical history of LF (N = 14/16). Fourteen of 21 patients (67%) with undetectable CD8+ T cell response had a measurable Lassa virus specific CD8+ T cell response with the 10-day assay. DISCUSSION: Despite reports of strong CD8+ T cell responses during acute Lassa virus infection, circulating Lassa virus-specific CD8+ T cells declined to undetectable levels in most Lassa fever survivors after ten years when evaluated with an 18-hour T cell stimulation. However, when Lassa virus-specific T cells were expanded prior to restimulation, a Lassa virus-specific CD8+ T cell response could be detected in many if the samples that were negative in the 18-hour stimulation assay, suggesting that prolonged cellular immunity does exist in Lassa fever survivors at low frequencies.

Predicting the evolution of the Lassa virus endemic area and population at risk over the next decades.

Lassa fever is a severe viral hemorrhagic fever caused by a zoonotic virus that repeatedly spills over to humans from its rodent reservoirs. It is currently not known how climate and land use changes could affect the endemic area of this virus, currently limited to parts of West Africa. By exploring the environmental data associated with virus occurrence using ecological niche modelling, we show how temperature, precipitation and the presence of pastures determine ecological suitability for virus circulation. Based on projections of climate, land use, and population changes, we find that regions in Central and East Africa will likely become suitable for Lassa virus over the next decades and estimate that the total population living in ecological conditions that are suitable for Lassa virus circulation may drastically increase by 2070. By analysing geotagged viral genomes using spatially-explicit phylogeography and simulating virus dispersal, we find that in the event of Lassa virus being introduced into a new suitable region, its spread might remain spatially limited over the first decades.

Cross-Reactive Antibodies to SARS-CoV-2 and MERS-CoV in Pre-COVID-19 Blood Samples from Sierra Leoneans.

Many countries in sub-Saharan Africa have experienced lower COVID-19 caseloads and fewer deaths than countries in other regions worldwide. Under-reporting of cases and a younger population could partly account for these differences, but pre-existing immunity to coronaviruses is another potential factor. Blood samples from Sierra Leonean Lassa fever and Ebola survivors and their contacts collected before the first reported COVID-19 cases were assessed using enzyme-linked immunosorbent assays for the presence of antibodies binding to proteins of coronaviruses that infect humans. Results were compared to COVID-19 subjects and healthy blood donors from the United States. Prior to the pandemic, Sierra Leoneans had more frequent exposures than Americans to coronaviruses with epitopes that cross-react with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), SARS-CoV, and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). The percentage of Sierra Leoneans with antibodies reacting to seasonal coronaviruses was also higher than for American blood donors. Serological responses to coronaviruses by Sierra Leoneans did not differ by age or sex. Approximately a quarter of Sierra Leonian pre-pandemic blood samples had neutralizing antibodies against SARS-CoV-2 pseudovirus, while about a third neutralized MERS-CoV pseudovirus. Prior exposures to coronaviruses that induce cross-protective immunity may contribute to reduced COVID-19 cases and deaths in Sierra Leone.

Space-Time Trends in Lassa Fever in Sierra Leone by ELISA Serostatus, 2012-2019.

Lassa fever (LF) is a viral hemorrhagic disease found in Sub-Saharan Africa and is responsible for up to 300,000 cases and 5000 deaths annually. LF is highly endemic in Sierra Leone, particularly in its Eastern Province. Kenema Government Hospital (KGH) maintains one of only a few LF isolation facilities in the world with year-round diagnostic testing. Here we focus on space-time trends for LF occurring in Sierra Leone between 2012 and 2019 to provide a current account of LF in the wake of the 2014-2016 Ebola epidemic. Data were analyzed for 3277 suspected LF cases and classified as acute, recent, and non-LF or prior LF exposure using enzyme-linked immunosorbent assays (ELISAs). Presentation rates for acute, recent, and non-LF or prior LF exposure were 6.0% (195/3277), 25.6% (838/3277), and 68.4% (2244/3277), respectively. Among 2051 non-LF or prior LF exposures, 33.2% (682/2051) tested positive for convalescent LF exposure. The overall LF case-fatality rate (CFR) was 78.5% (106/135). Both clinical presentations and confirmed LF cases declined following the Ebola epidemic. These declines coincided with an increased duration between illness onset and clinical presentation, perhaps suggesting more severe disease or presentation at later stages of illness. Acute LF cases and their corresponding CFRs peaked during the dry season (November to April). Subjects with recent (but not acute) LF exposure were more likely to present during the rainy season (May to October) than the dry season (p < 0.001). The findings here suggest that LF remains endemic in Sierra Leone and that caseloads are likely to resume at levels observed prior to the Ebola epidemic. The results provide insight on the current epidemiological profile of LF in Sierra Leone to facilitate LF vaccine studies and accentuate the need for LF cohort studies and continued advancements in LF diagnostics.

A Fc engineering approach to define functional humoral correlates of immunity against Ebola virus.

Protective Ebola virus (EBOV) antibodies have neutralizing activity and induction of antibody constant domain (Fc)-mediated innate immune effector functions. Efforts to enhance Fc effector functionality often focus on maximizing antibody-dependent cellular cytotoxicity, yet distinct combinations of functions could be critical for antibody-mediated protection. As neutralizing antibodies have been cloned from EBOV disease survivors, we sought to identify survivor Fc effector profiles to help guide Fc optimization strategies. Survivors developed a range of functional antibody responses, and we therefore applied a rapid, high-throughput Fc engineering platform to define the most protective profiles. We generated a library of Fc variants with identical antigen-binding fragments (Fabs) from an EBOV neutralizing antibody. Fc variants with antibody-mediated complement deposition and moderate natural killer (NK) cell activity demonstrated complete protective activity in a stringent in vivo mouse model. Our findings highlight the importance of specific effector functions in antibody-mediated protection, and the experimental platform presents a generalizable resource for identifying correlates of immunity to guide therapeutic antibody design.